The development of a vaccine against HIV-1 infection remains a significant global health problem. One of the major vaccine design concepts is the induction of high titer broadly neutralizing antibodies (bNAbs) that are capable of preventing HIV-1 entry into target cells, and hence intervening against virus transmission (Burton et al., Nat. Immunol., 5: 233-236 (2004)). A relevant target for bNAbs is the envelope, or Env, glycoprotein complex, or spike, on the virus surface (Zwick et al., Curr. HIV. Res., 5: 608-624 (2007); Walker et al., Curr. Opin. Immunol., 22: 358-366 (2010)). This complex, a trimer of gp120/gp41 heterodimers, attaches to receptors on target cells and then mediates fusion of the viral and cell membranes (Liu et al., Nature, 455: 109-113 (2008)). Any antibodies that can bind to a sufficient number of functional Env spikes will impede one or more stages in the receptor-binding and fusion process, thereby neutralizing infectivity (Zwick et al., Curr. HIV. Res., 5: 608-624 (2007)).
The development of vaccines which induce bNAbs remains elusive. One potential strategy is to create structural and antigenic mimics of the native, virion-associated Env spike. To do so a stop codon is inserted to truncate the gp41 protein prior to the membrane-spanning domain, thereby yielding soluble proteins that can be secreted from producer cells and purified. Unfortunately, without suitable sequence modifications, soluble gp140s are highly unstable and either disintegrate completely into their constituents or adopt non-native configurations that may be compromised from the perspective of bNAb induction (Guttman et al., J. Virol., 87: 11462-11475 (2013); Ringe et al., Proc. Natl. Acad. Sci. U.S.A., 110: 18256-18261 (2013)). One effective strategy for making soluble, native-like trimers involves introducing “SOSIP” modifications: (i) a disulfide bond (designated SOS) to link the gp120 and gp41 ectodomain (gp41ECTO) components, and (ii) a point substitution (I559P) in gp41ECTO that creates additional stability (Sanders et al., J. Virol., 76: 8875-8889 (2002); Binley et al., J. Virol., 74: 627-643 (2000); Binley et al., J. Virol., 76: 2606-2616 (2002)). Proteolytic cleavage between the gp120 and gp41 subunits is also critical for making native-like trimers, and is facilitated by optimizing the cleavage site and co-expressing the furin protease (Binley et al., J. Virol., 74: 627-643 (2000); Binley et al., J. Virol., 76: 2606-2616 (2002)). At present, the most advanced soluble, cleaved trimers, based on a subtype A founder virus, are designated BG505 SOSIP.664 (Sanders et al., PLoS Pathog., 9: e1003618 (2013); Wu et al., J. Virol., 80: 835-844 (2006); Hoffenberg et al., J. Virol., 87: 5372-5383 (2013)). These highly homogeneous trimers closely resemble virus spikes when visualized by negative stain electron microscopy (EM) (Sanders et al., PLoS Pathog., 9: e1003618 (2013)). They also have highly favorable antigenic properties, expressing multiple bNAb epitopes while occluding most epitopes for non-neutralizing antibodies (non-NAbs) (Ringe et al., Proc. Natl. Acad. Sci. U.S.A., 110: 18256-18261 (2013); Sanders et al., PLoSPathog., 9: e1003618 (2013)). In addition, the structure of these trimers has been determined by both x-ray diffraction and cryo-EM at ˜5-6 Å resolution (Julien et al., Science, 342: 1477-1483 (2013); Lyumkis et al., Science, 342: 1484-1490 (2013)).
A need remains for methods facilitating the production and manufacture of HIV Env glycoproteins (such as BG505 SOSIP.664 gp140 trimers), of sufficient quality, and in sufficient quantity, to serve as pre-clinical research reagents, and clinical-grade immunogens.